A fluorescent probe for detection of Hg2+ ions constructed by tetramethyl cucurbit[6]uril and 1,2-bis(4-pyridyl)ethene

In this paper, tetramethyl cucurbit[6]uril (TMeQ[6]) and 1,2-bis(4-pyridyl)ethene (G) were used to construct a supramolecular fluorescent probe G@TMeQ[6]. The host–guest interaction between TMeQ[6] and G was investigated using 1H NMR spectroscopy, single-crystal X-ray diffraction and various experimental techniques. The results show that TMeQ[6] and G form an inclusion complex with a host–guest ratio of 1:1 and the equilibrium association constant (Ka) was 2.494 × 104 M−1. The G@TMeQ[6] fluorescent probe can sensitively recognize Hg2+ ions by fluorescence enhancement. The linear range is 0.33 × 10−5–1.65 × 10−5 mol·L−1, R2 = 0.9926, and the limit of detection is 4.12 × 10−8 mol·L−1. The fluorescent probe can be used to detect the concentration of Hg2+ ions in aqueous solution, and provides a theoretical basis for the development of new fluorescent probes for detecting heavy metal ions.


Introduction
Mercury, as one of the most toxic heavy metal pollutants, not only seriously pollutes the ecological environment but also causes great harm to human health. Mercury and inorganic mercury ions (Hg 2+ ) in nature can be converted into organic mercury under the action of microorganisms, which cannot be decomposed or degraded into nontoxic substances in the human body [1,2], which will seriously threaten human health. Therefore, China has formulated water pollutant emission limits [3].
There is a π-π conjugation effect between the carbon-carbon double bond and the pyridine ring in 1,2-bis(4-pyridyl)ethene (G), which determines its ultraviolet absorption [39]. Because the N atom on the pyridine ring of the G molecule has lone-pair electrons, it can form coordination compounds with metal ions. At present, the host-guest fluorescent probes designed by G and Q[n]s have been rarely reported. Therefore, we constructed the host-guest fluorescent probes of TMeQ [6] and 1,2-bis(4pyridyl)ethene ( Figure 1). The fluorescence response and mechanism of metal ions were studied. It was found that G@TMeQ [6] had specific recognition of Hg 2+ ions in an aqueous solution, which provides a theoretical basis for the development of new fluorescent probes for the detection of heavy metal ions.

Results and Discussion
The interactions of G with TMeQ [6] UV-vis spectroscopy analysis The binding interaction between G and TMeQ [6] in aqueous solution was studied using UV-vis absorption spectroscopy. Figure 2a shows that the absorbance of G decreases with the addition of TMeQ [6], and the wavelength redshifts from 301 nm to 330 nm, indicating that TMeQ [6] has binding affinity for G. The molar ratio method (Figure 2b) shows that when n(TMeQ [6])/n(G) = 1:1, the absorption value of the system gradually stabilizes, indicating that the guest G and TMeQ [6] form an inclusion complex with a molar ratio of 1:1.

Fluorescent spectroscopic analysis
The interaction between G and TMeQ [6] in aqueous solution was investigated using fluorescence titration experiments. The fluorescence titration curve ( Figure 3a) shows that at the excitation wavelength of 351 nm, G has an emission peak at a wavelength of 350 nm. With the continuous addition of TMeQ [6], the fluorescence intensity of G is continuously enhanced, and the wavelength is redshifted to 391 nm, indicating that TMeQ [6] interacts with the guest molecule G. The TMeQ [6]    cavity may limit the rotation of the pyridine ring on the G molecule, and form an effective conjugated system with another pyridine ring outside the cavity, resulting in enhanced fluorescence [40]. The molar ratio curve ( Figure 3b) shows that when TMeQ [6] is added up to 1.0 equiv, the fluorescence intensity of the system gradually stabilizes, confirming that a G and TMeQ [6] inclusion complex is formed with a 1:1 stoichiometry.

Isothermal titration calorimetry (ITC) analysis
The association constant and thermodynamic parameters of the host-guest interaction between G and TMeQ [6] can be ob-tained using ITC. At 25 °C, a neutral aqueous solution of TMeQ [6] (1.0 × 10 −4 mol·L −1 , 1.00 mL) was gradually added to the aqueous solution of G, and the exothermic isotherms ( Figure S1 in Supporting Information File 1) and thermodynamic data (Table 1) were obtained. The equilibrium association constant (K a ) of G and TMeQ [6] is 2.494 × 10 4 M −1 , ΔH = −88.43 kJ/mol, which is an exothermic reaction (enthalpy-driven). The results show that the binding ability of G and TMeQ [6] is strong, and the ratio is 1:1. The results are consistent with those obtained by UV-vis spectroscopy and fluorescence spectroscopy.

Single-crystal X-ray diffraction analysis
The crystal structure of the inclusion complex formed by TMeQ [6] and G was obtained using X-ray single-crystal diffraction analysis. The crystal data and parameters are shown in Table 2. The single-crystal structure determination shows that the inclusion complex crystallizes in the triclinic crystal system, with the chiral space group P-1. Figure 4a shows that the basic crystal structure of complex 1 contains a TMeQ [6] molecule, a G molecule, a free water molecule and a [ZnCl 4 ] 2− anion. It can be clearly seen that one pyridyl group of the G molecule enters the cavity of TMeQ [6], whereas the other pyridyl group is outside the cavity, forming a 1:1 inclusion complex with TMeQ [6]. Figure 4b shows that the hydrogen atoms on the G molecule form C-H30···O1, C-H30···O2 and C-H31···O4 hydrogen bonds with the carbonyl oxygen and carbon atoms on TMeQ [6], and the bond distances are 2.163, 2.707 and 2.228 Å, respectively. In Figure 4c, the hydrogen atoms of G and the carbonyl oxygen of TMeQ [6] form C-H22···O1, C-H26···O1, C-H25···O4 and C-H27···O4 hydrogen bonds with bond distances of 2.370, 2.474, 2.564 and 2.685 Å, respectively. These interactions contribute to the formation of stable inclusion complexes. Figure 4d is a one-dimensional supramolecular chain of G@TMeQ [6], which is composed of hydrogen bonds C24-H···O6 and N13-H···O6 formed by the protons on the pyridyl group outside the cavity and the carbonyl oxygen of the adjacent TMeQ [6] port. The G molecule acts as a medium for connecting two adjacent TMeQ [6].
The possible mechanism of G@TMeQ [6] to detect Hg 2+ ions Fluorescence spectroscopy to investigate the specific recognition of Hg 2+ ions by G@TMeQ [6] The fluorescence response of G and G@TMeQ [6] to various metal ions (Fe 3+ , K + , Co 2+ , Mn 2+ , Cu 2+ , Ni 2+ , Cd 2+ , Zn 2+ , Pb 2+ , Cr 3+ , Cs + , Ca 2+ , Na + , Ba 2+ , Sr 2+ , Hg 2+ and other metal cations) in aqueous solution was investigated using fluorescence spectroscopy. It was found that the G molecule had no specific fluorescence response to the above metal cations (Figure 5a). Interestingly, the addition of the Hg 2+ ion to the G@TMeQ [6] system caused a strong fluorescence enhancement at an excitation wavelength of 351 nm (Figure 5b). The fluorescence intensity at the emission wavelength of 380 nm was enhanced from 25.8 to 831.2, indicating that the G@TMeQ [6] probe has a specific recognition of Hg 2+ .

Fluorescence spectroscopy to investigate the anti-interference and competitive ability of the probe to detect Hg 2+ ions
The metal cation interference experiment was used to investigate whether the G@TMeQ [6] fluorescent probe can selectively detect Hg 2+ ion in the presence of other metal cations ( Figure 6). The fluorescence intensity of the system was determined by adding other metal cations to the G@TMeQ[6]-Hg 2+ system. The results showed that in the presence of other metal  ions, the G@TMeQ [6] probe still showed specific recognition of Hg 2+ ions.

Fluorescence spectroscopy analysis of the interaction between the probe and Hg 2+ ions
The fluorescence titration curve of G@TMeQ [6] and Hg 2+ ions showed that the fluorescence intensity of the probe at 380 nm increased with the increase of Hg 2+ ion concentration (Figure 7a). When n(TMeQ [6])/n(G) = 1:1, increasing the concentration of Hg 2+ ion will not lead to a significant change in fluorescence intensity, indicating that the guest molecule G and TMeQ [6] form an inclusion complex with a molar ratio of 1:1. As shown in Figure 7b, when the concentration range of Hg 2+ ions is 0.33 × 10 -5 -1.65 × 10 -5 mol·L −1 , the fluorescence enhancement of the system has a good linear relationship with the concentration of Hg 2+ ion. The linear regression equation is y = 244.69x + 111.56, R 2 = 0.9926, and the detection limit (LOD = 3σ/K, here σ is the standard deviation and K is the slope of the calibration curve) is 4.12 × 10 -8 mol·L −1 . The G@TMeQ [6] fluorescent probe can effectively detect Hg 2+ ions in an aqueous solution.

H NMR spectroscopic titration analysis of the interaction between the probe and Hg 2+ ions
To study the solution complexation between G and TMeQ [6], the 1 H NMR titration spectrum of TMeQ [6] with different equivalents of guest molecule G was obtained ( Figure 8A). When 1.0 equiv G was added, it was found that the proton peak of the G molecule was split, and the chemical shift values of protons Ha′ and Hb′ were shifted upfield by 1.58 and 0.51 ppm, respectively, indicating that some protons on the G molecule entered the TMeQ [6] cavity and were shielded by the cavity. The chemical shift values of the protons Ha″, Hb″ and Hc′ shift downfield by 0.11, 0.62 and 0.50 ppm, respectively, indicating that the protons of this part are located at the port of the TMeQ [6], which is affected by the carbonyl oxygen of the TMeQ [6] port. When the G molecule is added to it, the free proton peak of the G molecule will be observed, indicating that  the G and TMeQ[6] form a 1:1 host-guest complex. The proton peaks on TMeQ [6] also split. The protons H1, H2 and H7 shift downfield by 0.11, 0.13 and 0.11 ppm, respectively, whereas the protons H3, H4, H5 and H6 shift upfield by 0.33, 0.15, 0.27 and 0.34 ppm, respectively. The above shows that the guest molecule partially enters the cavity of TMeQ [6].
The interaction mechanism between the fluorescent probe G@TMeQ [6] and Hg 2+ ion was studied using 1 H NMR titration experiments ( Figure 8B). After the addition of Hg 2+ ions to the G@TMeQ [6] system, the proton peaks Ha′, Hb′ and Hc′ on the guest molecules in the TMeQ [6] cavity move downfield until they disappear and become free guest proton peaks Ha, Hb and Hc, respectively. The protons Ha″ and Hb″ that did not enter the cavity also gradually moved downfield with the continuous addition of Hg 2+ ions. When 1.0 equiv. of Hg 2+ ions were added, they also became a free G molecule with proton peaks Ha, Hb. This indicates that Hg 2+ ions may coordinate with the port of TMeQ [6] to form a 1:1 coordination compound, which has a competitive effect with the G molecule. The G molecule wrapped in the cavity of the melon ring is squeezed out, and the proton peak of the G molecule will split, indicating that the TMeQ [6] also affects it.

Conclusion
We studied the host-guest interaction between G and TMeQ [6] and the specific recognition of Hg 2+ ions by the G@TMeQ [6] fluorescent probe. The interaction ratio of G to TMeQ [6] was 1:1 and the association constant was 2.494 × 10 4 M −1 . The interaction ratio between the G@TMeQ [6] probe and Hg 2+ was proved to be 1:1 using the molar ratio method and 1 H NMR titration spectroscopy. The recognition mechanism may be that there is a competitive effect between the Hg 2+ ion and the G molecule, which squeezes out part of G in the TMeQ [6] cavity and coordinates with the port of TMeQ [6]. The detection limit of probe G@TMeQ [6] for Hg 2+ ions is 4.12 × 10 −8 mol·L −1 . The fluorescent probe can effectively identify Hg 2+ ions and can be used for the detection of Hg 2+ ions in water.

Experimental
Materials 1,2-Bis(4-pyridyl)ethene (G) was purchased from Aladdin, and the other reagents were analytical grade and ready for use. TMeQ [6] was synthesized and purified in our laboratory.

Isothermal titration calorimetry (ITC)
The neutral aqueous solution of TMeQ [6] (1.0 × 10 -4 mol·L −1 , 1.00 mL) was placed in a sample tank, and G solution (1.0 × 10 -3 mol·L −1 ) was taken in a 250 μL syringe. The temperature was set at 25 °C, titrated 30 times (8 μL each time), and the titration time interval was 300 s. The thermodynamic parameters of each system were measured on a Nano ITC Isothermal Titration Calorimeter. After deleting the first unwanted data point, the data was analyzed using the independent model Launch Nano analysis software.

H NMR spectroscopy
The host-guest interaction between G and TMeQ [6] and the interaction mode between G@TMeQ[6] fluorescent probe and Hg 2+ ion were studied by 1 H NMR titration spectroscopy. All 1 H NMR spectroscopy data were recorded on a JEOL JNM-ECZ400s spectrometer in D 2 O at 293.15 K [42].

X-ray crystallography
Using single-crystal X-ray diffraction has been previously described in the literature [43]. The main crystal structure parameters are recorded in Table 2. In addition, CCDC-2225763 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/ data_request/cif.

Supporting Information
Supporting Information File 1